1. Field of the Invention
The invention relates generally to stem cells, and more specifically to a method for deriving endoderm cells using stem cells.
2. Background Information
During embryonic development, the tissues of the body are formed from three major cell populations: ectoderm, mesoderm and definitive endoderm. These cell populations, also known as primary germ cell layers, are formed through a process known as gastrulation. Following gastrulation, each primary germ cell layer generates a specific set of cell populations and tissues. Mesoderm gives rise to blood cells, endothelial cells, cardiac and skeletal muscle, and adipocytes. Definitive endoderm generates liver, pancreas and lung. Ectoderm gives rise to the nervous system, skin and adrenal tissues.
Human embryonic stem cells (ES) cells are pluripotent cells that can differentiate into a large array of cell types. When injected into immune-deficient mice, embryonic stem cells form differentiated tumors (teratomas). However, embryonic stem cells that are induced in vitro to form embryoid bodies (EBs) provide a source of embryonic stem cell lines that are amenable to differentiation into multiple cell types characteristic of several tissues under certain growth conditions. For example, ES cells become differentiated into neurons in the presence of nerve growth factor and retinoic acid.
Human ES cells and their differentiated progeny are important sources of normal human cells for therapeutic transplantation and for drug testing and development. Required by both of these goals is the provision of sufficient cells that are differentiated into tissue types suitable for a patient's needs or the appropriate pharmacological test. Associated with this is a need for an efficient and reliable method of producing differentiated cells from embryonic stem cells.
Currently, human embryonic stem cells (hES) are derived from three sources: blastocysts remaining after infertility treatments and donated for research, blastocysts generated from donated gametes (oocytes and sperm), and the products of nuclear transfer (NT). Cadaveric fetal tissue is the only source of human embryonic germ cells (hEG). hES and hEG cells offer remarkable scientific and therapeutic possibilities, involving potential for generating more specialized cells or tissues. Ethical concerns about the sources of hES and hEG cells, however, and fears that use of NT for research could lead to use of NT to produce a human being, have fostered a great deal of public discussion and debate.
Parthenogenic activation of mammalian oocytes may be used as an alternative to fertilization by sperm/NT to prepare oocytes for embryonic stem cell generation. Parthenogenic activation is the production of embryonic cells, with or without eventual development into an adult, from a female gamete in the absence of any contribution from a male gamete.
The first human parthenogenetic stem cells (hpSC) were derived from the inner cell mass of blastocysts obtained from unfertilized oocytes activated by chemical stimuli. These cells demonstrated characteristics typical for human embryonic stem cells (hESC), like extensive self-renewal and differentiation in vitro and in vivo into cells of all three germ layers. Human pSC that are histocompatible with significant segments of the human population due to the presence of homozygous HLA genotypes have been derived using an alternate oocyte activation technique (homozygous at all loci) or through the spontaneous activation of an oocyte of rare HLA homozygosity (heterozygous at most loci except HLA). These common HLA haplotype matched hpSC may reduce the risk of immune rejection after transplantation of their differentiated derivatives; thus offering significant advantages for application to cell-based therapies over hESC derived from fertilized oocytes having unique sets of HLA genes. Moreover, creation of hpSC overcomes the ethical hurdles associated with hESCs because the derivation of hpSC originates from unfertilized oocytes.
Two promising applications of pluripotent stem cells involve cell replacement therapy for diabetes or certain liver diseases associated with hepatocyte insufficiency. Production of high purity definitive endoderm (DE) is a critical first step in the generation of therapeutically useful cells of the DE lineage, such as hepatocytes and pancreatic endocrine cells.
Definitive endoderm is formed during gastrulation along with the two other principal germ layers—ectoderm and mesoderm, and during development will give rise to the gastrointestinal and respiratory tracts as well as other organs including the liver and pancreas. The efficient generation of DE from hESC requires two conditions: signaling by transforming growth factor β family members such as Activin A or Nodal as well as release from pluripotent self-renewal signals generated by insulin/insulin-like growth factor signaling via phosphatidylinositol 3-kinase (PI3K). Moreover, adding Wnt3a together with the Activin A increases the efficiency of mesendoderm specification, a bipotential precursor of DE and mesoderm, and improves the synchrony with which the hESCs are initiated down the path toward DE formation.
The developmental capacity of hESC-derived DE has been demonstrated both in vitro and in vivo. Various hESC differentiation protocols utilizing as a first stage differentiation to enriched populations of DE have resulted in production of hepatocyte-like cells exhibiting some characteristics of mature hepatocytes or production of islet endocrine-like cells capable of synthesizing the pancreatic hormones. Transplantation of the hESC-derived DE cells under the kidney capsule of severe combined immunodeficient (SCID) mice resulted in their differentiation into more mature cells of endodermal organs expressing CDX2, villin and hepatocyte-specific antigen. In a mouse model of acute liver injury, the hESC-derived DE further differentiated into hepatocytes was shown to repopulate the damaged liver. Moreover, it was shown that pancreatic endoderm cells differentiated from hESC-derived DE developed in vivo into glucose-responsive endocrine cells that are morphologically and functionally similar to pancreatic islets and protect mice against streptozotocin—induced hyperglycemia.
Numerous studies have contributed to the understanding of global gene expression patterns in pluripotent stem cells and variations therein may partially determine the capacity for differentiation. The control of gene expression is in part regulated by epigenetic mechanisms, including post-translational modifications of histones and DNA methylation. Molecular instruments that disrupt global epigenetic mechanisms may play a role in the elucidation of genetic circuits operating in stem cells. One candidate for global epigenetic modulation is the pharmaceutical agent TSA, a potent histone deacetylase inhibitor. It was shown that TSA treatment of mouse embryonic stem cells causes suppression of important pluripotency factors, including Nanog, a master regulator of stem cell identity, and the activation of differentiation related genes. Interesting, in that study, TSA effect did not support the maintenance or progression of differentiation; upon removal of TSA, the cells reverted to the undifferentiated phenotype.
Provided herein is a method for the differentiation of stem cells to definitive endoderm cells that produces highly enriched cultures of differentiated cells.